Phospholipase D (PLD; EC3.1.4.4.) enzymes are phosphodiesterases that hydrolyze phospholipids to phosphatidic acid (PA) are their free head groups. In mammalian cells the principal substrate is phosphatidylcholine (PC), and the production of PA has broad biological functions. PA regulates biophysical properties of cellular membranes, acts as a second messenger to alter activities of many enzymes and proteins, and is subsequently metabolized to diacylglycerols and lysophosphatidic acids by lipid phosphate phosphatase and phospholipase A2, respectively. Diacylglycerols derived from PCs are important cellular signaling molecules and lysophosphatidic acid is released as an extracellular messenger that affects many cell types. Evidence supports a role for PLD in regulated exocytosis, cell proliferation, membrane trafficking, and tumor formation.
Isoenzymes of PLD have been cloned from animals, fungi, plants, bacteria, and viruses. Two mammalian PLD genes (PLD1 and PLD2) have been identified, and several splice variant protein products have been characterized. The mammalian isoenzymes have a conserved primary sequence and domain structure but are differentially regulated by upstream signaling pathways. Both enzymes are members of the PXPH-PLD subfamily that have a pleckstrin homology (PH) and phox homology (PX) domains in tandem at their N terminal (Eliás M, Potocký M, Cvrcková F, Zárskjý V., “Molecular diversity of phospholipase D in angiosperms,” BMC Genomics. 2002; 3(1):2. Epub 2002 Feb. 1) and are hypothesized to have pseudodimeric catalytic domains with invariant HXKX4D motifs (Ponting C P, Kerr I D., “A novel family of phospholipase D homologues that includes phospholipid synthases and putative endonucleases: identification of duplicated repeats and potential active site residues,” Protein Sci. 1996 May; 5(5):914-22.; Stuckey J. A., Dixon J. E., “Crystal structure of a phospholipase D family member,” Nat Struct Biol. 1999 March; 6(3):278-84.).
The production of PA has broad physiological impact and choline release has been suggested to play a role in acetylcholine synthesis. PLD was initially identified in plants and early experiments on purified cabbage PLD (Yang S. F., Freer S., Benson A. A., “Transphosphatidylation by phospholipase D,” J Biol Chem. 1967 Feb. 10; 242(3):477-84.) established both a phosphatidylcholine phosphohydrolase activity (Eq. 1) and a competing phosphatidylcholine transphosphatidylase activity (Eq. 2).Phosphatidylcholine+H20→phosphatidic acid+choline  (Eq. 1)Phosphatidylcholine+ROH→phosphatidyl-OR+choline  (Eq. 2)
Transfer of phosphatidyl groups from phospholipid substrates to primary alcohols (ROH) yields phosphatidylalcohols (Eq. 2). Because of their unique origin, low abundance in biological membranes, and metabolic stability, the formation of phosphatidylalcohols has been used as a specific marker for PLD activity. Specific in vitro and in vivo assay systems have been developed to allow the identification of signaling modulators, relative activities, and substrate-product relationships using electrospray ionization mass spectrometry (Brown, H. A., Gutowski, S., Moomaw, C. R., Slaughter, C., and Sternweis, P. C. (1993) ADP-ribosylation factor (ARF), a small GTP-dependent regulatory protein, stimulates phospholipase D activity. Cell 75: 1137-1144; Walker, S. J. and Brown, H. A. (2003) Measurement of G protein stimulated phospholipase D activity in intact cells. In Methods in Molecular Biology vol 237: G Protein Signaling: Methods and Protocols, edited by A.V. Smrcka.Humana Press Inc., Totowa, N.J.; and Brown, H. A., Henage, L. G., Preininger, A. M., Xiang, Y., and Exton, J. H. (2007) Biochemical Analysis of Phospholipase D. In Methods in Enzymology. 434, Lipidomics and Bioactive Lipids: Lipids and Cell Signaling. Edited by H. Alex Brown. Elsevier. pp. 49-87.).
Because of the roles of PLD and its products, the enzymatic activity of PLD is tightly regulated by a variety of hormones, neurotransmitters, growth factors, cytokines, integrins, and other cellular signals. The PLD1 isoenzyme is under extensive control both in vitro and in vivo. This tight regulation is in part the reason the enzyme was difficult to study for several decades until it was determined that phosphatidylinositol 4,5-bisphosphate (PIN was an essential regulator of catalytic activity (Brown et al., 1993). Interaction between PLD and lipid vesicles is dependent upon PIP2 (Henage, L., Exton, J. and Brown, H. A. Kinetic analysis of a mammalian Phospholipase D: Kinetic analysis of a mammalian Phospholipase D: Allosteric modulation by monomeric GTPases, Protein kinase C and polyphosphoinosites. (2006) J. Biol. Chem. 281: 3408-3417.). This interaction is mediated primarily by a conserved polybasic region within a C-terminal PLD catalytic subdomain, but there also appear to be additional sites where PLD and phosphoinositides directly interact. Previous work suggests that there are distinct yet interacting binding sites for the major regulators. Mutational studies have identified PLD1 domains and amino acid sequences involved in these interactions. PLD1 activity is regulated by conventional isoforms of protein kinase C through a direct protein-protein interaction that is not dependent upon kinase activity. PLD1 is also regulated by members of the Rho and Arf GTPase families.
By contrast PLD2 has a relatively high basal activity and does not require modulation by GTPases for activation. Initially this lead to an incorrect assumption that PLD1 was the signaling isoenzyme and PLD2 was involved in more mundane housekeeping functions. Recent evidence has shown that both PLD1 and PLD2 are activated by many cell surface receptors, including tyrosine kinase growth factor receptors. The production of PA appears to be essential to mediating the downstream processes modulated by these growth promoting and cell proliferative pathways.
Previous work from a number of laboratories has suggested a role for PLD in a number of cellular processes required for growth, proliferation, transformation and tumor formation. The presence of Arf and PLD on Golgi membranes followed by reports that PA has a role in the formation of coated vesicles suggests a role in vesicle trafficking. PLD is associated with the translocation of the glucose transporter, Glut-4, to the plasma membrane and PLD activity has been implicated in the internalization of the epidermal growth factor receptor. These studies have relied on the use of primary alcohols to interfere with the formation of PA by PLD, but further support has been obtained through the expression of catalytically inactive PLD mutants and more recently using RNA interference techniques to block the expression of PLD isoenzymes. Each of these approaches has limitations, so the development of specific, isoenzyme selective inhibitors is expected to greatly complement these previous approaches. PLD couples signal-transduction networks bidirectionally to the cytoskeleton. Several findings have implicated a role for PLD in controlling cell shape, motility, chemotaxis, and vesicle trafficking. Rho, Rac, and Cdc42 have been implicated in these processes as well and each has been shown to activate PLD through protein-protein interactions.
Many studies have implicated a role for PLD in regulation of cell survival. Many signaling networks and mitogenic signals involved in modulating cell survival and apoptotic pathways have been shown to include a role for PLD. The specifics vary with cell types. In some cells PLD activity is proapoptotic, while in others PLD promotes cell survival and mitogenesis. Recent findings suggest that PLD1 activation leads to an increase in RAS-ERK/PI3K (reviewed in Andresen B T, Rizzo M A, Shome K, Romero G., “The role of phosphatidic acid in the regulation of the Ras/MEK/Erk signaling cascade,” FEBS Lett. 2002 Oct. 30; 531(1):65-8. Review.) and NFκB signaling cascade (Dong Woo Kang, Mi Hee Park, Craig Lindsley, H Alex Brown and Do Sik Min, “Regulation of Phospholipase D1 signaling dynamics via enzymatic activity dependent positive feedback loop,” Molecular and Cellular Biology (2009), in review.) and subsequently selective expression of PLD1 in breast cancer cells. This leads to growth factor-induced matrix metalloproteinase upregulation which is essential to cancer cell migration and metastasis. PLD generated PA has also been proposed to directly modulated the mTOR pathway, which is known to play a major role in the development of several types of human cancers (reviewed by Foster D A. “Regulation of mTOR by phosphatidic acid?” Cancer Res. 2007 Jan. 1; 67(1):1-4. Review; Sun Y, Chen J. “mTOR signaling: PLD takes center stage,” Cell Cycle. 2008 October; 7(20):3118-23. Epub 2008 Oct. 27. Review.). Growth factor receptor signaling pathways, information on cell growth, nutrient status, and cellular bioenergetics are integrated through the mTOR circuit. Several recent studies suggest that the actions of many cellular oncogenes may explain the pioneering observations of Otto Warburg with regard to the propensity of most cancer cells to preferentially utilize aerobic glycolysis pathway in their cellular bioenergetics. Several recent studies have suggested that modulation of the mTOR pathway may be among the most important with respect to the development of the next generation of anti-cancer therapeutics (Vander Heiden M G, Cantley L C, Thompson C B., “Understanding the Warburg effect: the metabolic requirements of cell proliferation,” Science. 2009 May 22; 324(5930):1029-33.). PLD appears to play an important role in the regulation of these key signaling and cell survival pathways. Isoenzyme selective inhibitors of PLD may provide a novel and effective mechanism for inhibition of cancer cell transformation and metastasis.
Despite previous research, there remains a need for compounds and compositions useful as isoform selective Phospholipase D inhibitors that overcome current deficiencies and that effectively treat diseases and disorders associated with Phospholipase D. The disclosed compounds, compositions, and methods address this need as well as other needs.